Recent in vivo studies from several laboratories have challenged the paradigm that tissue factor is solely responsible for triggering blood clotting in thrombotic disease, by showing that blocking the function of coagulation factor XII (which is essential for the contact pathway of blood clotting) significantly decreases thrombosis in animal models. However, for many years it has been unclear how the contact pathway is initiated in vivo. Recent work from our laboratory has identified inorganic polyphosphate as the long-sought (patho) physiologic activator of the contact pathway. Polyphosphate is secreted from activated platelets and accumulates in infectious microorganisms; we propose that it is an important triggering agent at the nexus of prothrombotic and pro-inflammatory pathways. Our studies have now shown that, in addition to being an extremely potent initiator of the contact pathway, polyphosphate also accelerates factor V activation and enhances fibrin clot structure, leading to thicker fibrin fibrils that are more resistant to fibrinolysis. Because the ability of polyphosphate to modulate blood clotting has only recently been discovered, we still do not understand mechanistically how polyphosphate exerts its effects on coagulation. Similarly, many questions remain regarding where, when and how if functions in vivo. These questions will be addressed in three aims, which focus on: understanding how polyphosphate triggers the contact pathway of clotting; understanding how polyphosphate accelerates factor V activation; and understanding how polyphosphate functions in vivo. The work outlined in this grant proposal will therefore provide a mechanistic understanding of how polyphosphate functions in hemostasis, thrombosis and inflammation. These studies are designed to identify novel drug targets for interrupting thrombotic and inflammatory pathways with minimized risk of bleeding side effects. They are also designed to develop novel hemostatic agents for treating bleeding.